Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N35/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical
  • A01N35/02—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical containing aliphatically bound aldehyde or keto groups, or thio analogues thereof; Derivatives thereof, e.g. acetals
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N33/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic nitrogen compounds
  • A01N33/02—Amines; Quaternary ammonium compounds
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N31/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic oxygen or sulfur compounds
  • A01N31/02—Acyclic compounds
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N31/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic oxygen or sulfur compounds
  • A01N31/04—Oxygen or sulfur attached to an aliphatic side-chain of a carbocyclic ring system
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N35/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical
  • A01N35/04—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical containing aldehyde or keto groups, or thio analogues thereof, directly attached to an aromatic ring system, e.g. acetophenone; Derivatives thereof, e.g. acetals
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
  • A01N37/06—Unsaturated carboxylic acids or thio analogues thereof; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
  • A01N37/08—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing carboxylic groups or thio analogues thereof, directly attached by the carbon atom to a cycloaliphatic ring; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
  • A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
  • A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
  • A01N43/06—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom five-membered rings
  • A01N43/12—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom five-membered rings condensed with a carbocyclic ring
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
  • A01P13/00—Herbicides; Algicides
  • A01P13/02—Herbicides; Algicides selective
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
  • A01P21/00—Plant growth regulators

Definitions

• This disclosure relates to methods for regulating growth in a plant. Specifically, the methods of the disclosure regulate the growth of the plant by administration of a composition comprising an effective amount of one or more apocarotenoids to the plant, its part, or the seed.
• Apocarotenoids secondary metabolites derived from carotenoids such as beta- carotene
• strigolactone and absisic acid are apocarotenoids important for plant root and shoot structure.
• One particularly important class of apocarotenoids in vertebrate development consists of retinoids, specifically retinal, retinol, and retinoic acid.
• retinoids specifically retinal, retinol, and retinoic acid.
• Proper levels of beta-carotene-derived retinoids are critical during human embryogenesis.
• Retinoid deficiency causes morphological defects and blindness. Excess retinoids can lead to physical and cognitive birth defects.
• Retinoid binding proteins (RBPs) have diverse roles in many developmental processes, including
• Rhodopsin a light-sensitive complex composed of an opsin protein bound to retinal, is crucial for photoreception in a range of animal, bacteria, and algae species.
• Rhodopsin a light-sensitive complex composed of an opsin protein bound to retinal.
• the function of retinoids in multi-cellular development outside of the animal kingdom has remained uncharacterized.
• Natural signaling molecules capable of increasing root depth and branching are highly desirable tools for enhancing stress tolerance in crops.
• the inventors have found novel modulators of root architecture in plants when applied exogenously to the plants.
• beta-carotene-derived metabolites e.g ., apocarotenoids
• apocarotenoids can act as plant growth regulators by modifying root architecture, in particular the depth and density of a root system, when applied exogenously to a plant, and methods of making and using said compounds.
• one aspect of the disclosure provides methods for regulating growth in a plant, the method comprising exogenously contacting a composition comprising an effective amount of one or more apocarotenoids to the plant, a plant part, or a plant seed.
• Another aspect of the disclosure provides methods for improving drought tolerance in a plant, the method comprising exogenously contacting a composition comprising an effective amount of one or more apocarotenoids to the plant, a plant part, or a plant seed.
• Another aspect of the disclosure provides methods for fertilizing soil, the method comprising providing a composition comprising an effective amount of one or more apocarotenoids to the soil.
• Another aspect of the disclosure provides a method for controlling a harmful plant in a crop, the method comprising exogenously contacting a composition comprising a herbicidally active amount of one or more apocarotenoids to the harmful plant, a harmful plant part, a harmful plant seed and/or an area in which the harmful plant grows.
• Another aspect of the disclosure provides a method for identifying a plant protein associated with a root development phenotype, the method comprising identifying a plant protein that has significant homology to a vertebrate retinoid binding protein, and
• compositions comprising, consisting of, or consisting essentially of an effective amount of one or more apocarotenoids present in the composition in a concentration of about 0.01 mM to about 100 mM and an agriculturally acceptable carrier, excipient, and/or diluent.
• FIG. 1A-1E illustrates a reporter for RA binding proteins highlighting distinct developmental regions in th e Arabidopsis root.
• FIG. 1A is a schematic showing how plant roots can be divided into spatially distinct regions: the meristem zone composed of pluripotent mitotic cells, the cell elongation zone, and the differentiation zone.
• FIG. IB is a schematic showing that merocyanine aldehyde is a reporter that only becomes fluorescent when bound to the active site of an RA binding protein.
• FIG. 1C are fluorescent images showing that Arabidopsis roots treated with the merocyanine aldehyde reporter exhibit dynamic fluorescence in the meristem (M), elongation (E), and differentiation (D) zones.
• FIG. ID is a graph showing cells at the center of the bright merocyanine aldehyde pulses in the elongation and differentiation zones are significantly more likely to be close to the sites of lateral root primordia (LRP) than control cells.
• FIG. IE is a graph showing that D15, a known inhibitor of lateral root organogenesis, reduces the number of dynamic fluorescent pulses of merocyanine.
• FIG. 2A-2B illustrates that endogenous beta-carotene-derived apocarotenals can be identified in whole plant tissue.
• FIG. 2A shows structures of hydrophobic carotenals (ApolO, Apol2, Apol4, and Retinal) and comparatively hydrophilic carotenals (Beta-ionone and Beta-cyclocitral).
• FIG. 2B is a graph showing changes in whole plant levels of
• FIG. 3 shows fluorescence images demonstrating that application of apocarotenals increase the oscillation zone maxima in Dl5-treated plants.
• FIG. 4A-4D illustrates the effects of exogenously applied apocarotenoids on root development.
• FIG. 4A is a graph showing the effects of 10 mM of select apocarotenoids (psuedoinonone (PI), dihydroactinidiolide (DHAD), abscisic acid (ABA), ApolO, Apol2, Apol4, and retinal) on the oscillation amplitude of the lateral root clock.
• FIG. 4B are graphs showing the effects of co-treatment with 500 nM Apol4 and D15 on primary root growth (left) and Apol4 rescue of lateral root capacity in D15 treated plants, normalized to the control treatment (right).
• FIG. 4C are graphs showing the effects of co-treatment with 1 pM retinal and 125 pM D15 on primary root growth (left) and lateral root branching (right).
• FIG. 4D is a graph showing the effects of exogenous retinal on lateral root primordia.
• FIG. 5 is a graph showing plants with mutations in putative plant RBP-encoding genes have defects in their capacity to produce lateral roots.
• FIG. 6 shows fluorescent images showing merocyanine aldehyde fluorescence in maize, cucumber, sunflower, rice, and carrot roots.
• Ranges can be expressed herein as from“about” one particular value, and/or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
• the term“about” in association with a numerical value means that the numerical value can vary plus or minus by 5% or less of the numerical value.
• the term“contacting” includes the physical contact of at least one substance to another substance.
• alkenyl as used herein, means a straight or branched chain hydrocarbon containing from 2 to 10 carbons, unless otherwise specified, and containing at least one carbon-carbon double bond.
• alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-l -heptenyl, 3-decenyl, and 3,7-dimethylocta-2,6-dienyl.
• alkoxy means an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom.
• Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert- butoxy, pentyloxy, and hexyloxy.
• alkyl as used herein, means a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms unless otherwise specified.
• Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2- dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.
• an“alkyl” group is a linking group between two other moieties, then it may also be a straight or branched chain; examples include, but are not limited to -CH 2 -, -CH2CH2-, - CH 2 CH 2 CHC(CH 3 )-, -CH 2 CH(CH 2 CH 3 )CH 2 _.
• alkylene refers to a bivalent alkyl group.
• An“alkylene chain” is a polymethylene group, i.e., -(CH 2 )n-, wherein n is a positive integer, preferably from one to six, from one to four, from one to three, from one to two, or from two to three.
• a substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms is replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group.
• An alkylene chain also may be substituted at one or more positions with an aliphatic group or a substituted aliphatic group.
• alkynyl as used herein, means a straight or branched chain hydrocarbon group containing from 2 to 10 carbon atoms and containing at least one carbon-carbon triple bond.
• Representative examples of alkynyl include, but are not limited, to acetylenyl, 1 - propynyl, 2-propynyl, 3-butynyl, 2-pentynyl, and l-butynyl.
• aryl means a phenyl (i.e., monocyclic aryl), or a bicyclic ring system containing at least one phenyl ring or an aromatic bicyclic ring containing only carbon atoms in the aromatic bicyclic ring system.
• the bicyclic aryl can be azulenyl, naphthyl, or a phenyl fused to a monocyclic cycloalkyl, a monocyclic cycloalkenyl, or a monocyclic heterocyclyl.
• the bicyclic aryl is attached to the parent molecular moiety through any carbon atom contained within the phenyl portion of the bicyclic system, or any carbon atom with the napthyl or azulenyl ring.
• the fused monocyclic cycloalkyl or monocyclic heterocyclyl portions of the bicyclic aryl are optionally substituted with one or two oxo and/or thia groups.
• Representative examples of the bicyclic aryls include, but are not limited to, azulenyl, naphthyl, dihydroinden-l -yl, di ydroinden-2-yl, dihydroinden-3-yl,
• dihydroinden-4-yl 2,3-dihydroindol-4-yl, 2,3-dihydroindol-5-yl, 2,3-di ydroindol-6-yl, 2,3- dihydroindol-7-yl, inden-l -yl, inden-2-yl, inden-3-yl, inden-4-yl, dihydronaphthalen-2-yl, dihydronaphthalen-3-yl, dihydronaphthalen-4-yl, dihydronaphthalen-l -yl, 5, 6, 7, 8- tetrahydronaphthalen-l-yl, 5,6,7,8-tetrahydronaphthalen-2-yl, 2,3-dihydrobenzofuran-4-yl, 2,3-dihydrobenzofuran-5-yl, 2,3-dihydrobenzofuran-6-yl, 2,3-dihydrobenzofuran-7-yl,
• the bicyclic aryl is (i) naphthyl or (ii) a phenyl ring fused to either a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, or a 5 or 6 membered monocyclic heterocyclyl, wherein the fused cycloalkyl, cycloalkenyl, and heterocyclyl groups are optionally substituted with one or two groups which are
• An“aralkyl” or“arylalkyl” group comprises an aryl group covalently attached to an alkyl group, either of which independently is optionally substituted.
• the aralkyl group is aryl(Cl-C6)alkyl, including, without limitation, benzyl, phenethyl, and
• cycloalkyl as used herein, means a monocyclic or a bicyclic cycloalkyl ring system.
• Monocyclic ring systems are cyclic hydrocarbon groups containing from 3 to 8 carbon atoms, where such groups can be saturated or unsaturated, but not aromatic. In certain embodiments, cycloalkyl groups are fully saturated. Examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.
• Bicyclic cycloalkyl ring systems are bridged monocyclic rings or fused bicyclic rings.
• Bridged monocyclic rings contain a monocyclic cycloalkyl ring where two non-adjacent carbon atoms of the monocyclic ring are linked by an alkylene bridge of between one and three additional carbon atoms (i.e. , a bridging group of the form -(CH 2 )W-, where w is 1 , 2, or 3).
• Representative examples of bicyclic ring systems include, but are not limited to, bicyclo[3.l .ljheptane, bicyclo[2.2.l Jheptane, bicyclo[2.2.2]octane,
• Fused bicyclic cycloalkyl ring systems contain a monocyclic cycloalkyl ring fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl, or a monocyclic heteroaryl.
• the bridged or fused bicyclic cycloalkyl is attached to the parent molecular moiety through any carbon atom contained within the monocyclic cycloalkyl ring.
• Cycloalkyl groups are optionally substituted with one or two groups which are independently oxo or thia.
• the fused bicyclic cycloalkyl is a 5 or 6 membered monocyclic cycloalkyl ring fused to either a phenyl ring, a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, a 5 or 6 membered monocyclic heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl, wherein the fused bicyclic cycloalkyl is optionally substituted by one or two groups which are independently oxo or thia.
• halo or“halogen” as used herein, means -Cl, -Br, -I or -F.
• haloaliphatic “haloalkyl,”“haloalkenyl,” and“haloalkoxy” refer to an aliphatic, alkyl, alkenyl or alkoxy group, as the case may be, which is substituted with one or more halogen atoms.
• heteroaryl means a monocyclic heteroaryl or a bicyclic ring system containing at least one heteroaromatic ring.
• the monocyclic heteroaryl can be a 5 or 6 membered ring.
• the 5 membered ring consists of two double bonds and one, two, three or four nitrogen atoms and optionally one oxygen or sulfur atom.
• the 6 membered ring consists of three double bonds and one, two, three or four nitrogen atoms.
• the 5 or 6 membered heteroaryl is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the heteroaryl.
• monocyclic heteroaryl include, but are not limited to, furyl, imidazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, oxazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyrrolyl, tetrazolyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, and triazinyl.
• the bicyclic heteroaryl consists of a monocyclic heteroaryl fused to a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl, or a monocyclic heteroaryl.
• the fused cycloalkyl or heterocyclyl portion of the bicyclic heteroaryl group is optionally substituted with one or two groups which are independently oxo or thia.
• the bicyclic heteroaryl contains a fused cycloalkyl, cycloalkenyl, or heterocyclyl ring
• the bicyclic heteroaryl group is connected to the parent molecular moiety through any carbon or nitrogen atom contained within the monocyclic heteroaryl portion of the bicyclic ring system.
• the bicyclic heteroaryl is a monocyclic heteroaryl fused to a benzo ring
• the bicyclic heteroaryl group is connected to the parent molecular moiety through any carbon atom or nitrogen atom within the bicyclic ring system.
• bicyclic heteroaryl include, but are not limited to, benzimidazolyl, benzofuranyl, benzothienyl, benzoxadiazolyl, benzoxathiadiazolyl, benzothiazolyl, cinnolinyl, 5,6-dihydroquinolin-2-yl, 5,6-dihydroisoquinolin-l-yl, furopyridinyl, indazolyl, indolyl, isoquinolinyl, naphthyridinyl, quinolinyl, purinyl, 5, 6,7,8- tetrahydroquinolin- 2-yl, 5,6,7,8-tetrahydroquinolin-3-yl, 5,6,7,8-tetrahydroquinolin-4-yl, 5,6,7,8-tetrahydroisoquinolin-l -yl, thienopyridinyl, 4,5,6,7-tetrahydrobenz
• heterocyclyl and“heterocycloalkyl” as used herein, mean a monocyclic heterocycle or a bicyclic heterocycle.
• the monocyclic heterocycle is a 3, 4, 5, 6 or 7 membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S where the ring is saturated or unsaturated, but not aromatic.
• the 3 or 4 membered ring contains 1 heteroatom selected from the group consisting of O, N and S.
• the 5 membered ring can contain zero or one double bond and one, two or three heteroatoms selected from the group consisting of O, N and S.
• the 6 or 7 membered ring contains zero, one or two double bonds and one, two or three heteroatoms selected from the group consisting of O, N and S.
• the monocyclic heterocycle is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the monocyclic heterocycle.
• monocyclic heterocycle include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1 ,3-dioxanyl, 1 ,3-dioxolanyl, 1 ,3- dithiolanyl, 1 ,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothien
• the bicyclic heterocycle is a monocyclic heterocycle fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocycle, or a monocyclic heteroaryl.
• the bicyclic heterocycle is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the monocyclic heterocycle portion of the bicyclic ring system.
• bicyclic heterocyclyls include, but are not limited to, 2,3-dihydrobenzofuran-2-yl, 2,3- dihydrobenzofuran-3-yl, indolin-l -yl, indolin-2-yl, indolin-3-yl, 2,3-dihydrobenzothien-2-yl, decahydroquinolinyl, decahydroisoquinolinyl, octahydro-l H-indolyl, and
• Heterocyclyl groups are optionally substituted with one or two groups which are independently oxo or thia.
• heterocyclyl is a 5 or 6 membered monocyclic heterocyclyl ring fused to phenyl ring, a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, a 5 or 6 membered monocyclic heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl, wherein the bicyclic heterocyclyl is optionally substituted by one or two groups which are
• saturated means the referenced chemical structure does not contain any multiple carbon-carbon bonds.
• a saturated cycloalkyl group as defined herein includes cyclohexyl, cyclopropyl, and the like.
• substituted means that a hydrogen radical of the designated moiety is replaced with the radical of a specified substituent, provided that the substitution results in a stable or chemically feasible compound.
• substituted when used in reference to a designated atom, means that attached to the atom is a hydrogen radical, which can be replaced with the radical of a suitable substituent.
• substituents refers to a number of substituents that equals from one to the maximum number of substituents possible based on the number of available bonding sites, provided that the above conditions of stability and chemical feasibility are met.
• an optionally substituted group may have a substituent at each substitutable position of the group, and the substituents may be either the same or different.
• the term“independently selected” means that the same or different values may be selected for multiple instances of a given variable in a single compound.
• the term“unsaturated” as used herein means the referenced chemical structure contains at least one multiple carbon-carbon bond, but is not aromatic.
• an unsaturated cycloalkyl group as defined herein includes cyclohexenyl, cyclopentenyl, cyclohexadienyl, and the like.
• compositions comprising compounds (e.g., apocarotenoids) that act as plant growth regulators. These compounds can modify root architecture, which plays a critical role in a plant’s ability to respond to diverse
• the term“effective amount” refers to that amount of the
• an“effective amount” comprises a low concentration (e.g., in the nM or mM concentration range) of the composition.
• the amounts of the composition (and any additional compounds) which are optimal in each case depend on the nature of the apocarotenoid used and any additional compounds used and on the nature and development stage of the plant stock to be treated, and can be determined in each individual case by simple, routine preliminary tests.
• the effective amount of one or more apocarotenoids is at least about 100 nM per m 3 of soil, or at least about 10 mg per m 3 of soil.
• the term“herbicidally active amount” in the context of the present disclosure means an amount of the composition that is suitable for adversely affecting plant growth (e.g., reducing drought tolerance, killing the plant, etc.).
• a “herbicidally active amount” comprises a high concentration (e.g., 100 mM or above or from about 100 pM to about 100 mM) of the agricultural compound.
• root architecture means the spatial arrangement of a plant’s root tissue within the soil.
• the term“improving growth” refers to promoting, increasing or improving the rate of growth of the plant or increasing or promoting an increase in the size of the plant.
• the term“reducing growth” refers to decreasing or slowing the rate of growth of the plant or decreasing or promoting a decrease in the size of the plant.
• All percentages, ratios and proportions herein are by weight, unless otherwise specified. A weight percent (weight %, also as wt %) of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included (e.g., on the total amount of an apocarotenoid).
• the methods and compositions described herein can be configured by the person of ordinary skill in the art to meet the desired need.
• the disclosed materials, methods, and apparati provide apocarotenoids that can modify root architecture when applied exogenously to plants.
• Root architecture plays a major role in plant fitness, crop performance, and/or grain yield.
• the inventors have found novel regulators of root architecture in plants when applied exogenously to the plants. For example, low concentrations (e.g., in the nM or mM concentration range) of apocarotenoids of the disclosure (e.g., Apol4, retinal) were found to fully rescue root growth and lateral root organogenesis in the presence of an apocarotenoid synthesis inhibitor. Lateral roots constitute the majority of a mature plant’s root system and are vital for plant anchorage, absorption of water and nutrients, and survival in the presence of diverse environmental stresses such as drought.
• compositions comprising, consisting of, or consisting essentially of one or more apocarotenoids.
• apocarotenoid means a cleavage product derived from one or more carotenoids. Included within the term“apocarotenoid” are“apocarotenals,” which include those carotenoids that contain an aldehyde functional group. Most
• apocarotenoids are normally present at very low levels in the roots of plants.
• Apocarotenoids are formed by the action of nine different carotenoid cleavage dioxygenases (CCDs).
• CCDs carotenoid cleavage dioxygenases
• the specific products generated by each individual CCD are difficult to characterize because CCDs seem to have functional redundancy.
• D15 N- (4-fluorobenzyl)-methoxy cinnamic hydroxamic acid
• ccd8 mutants do not have reduced lateral root capacity. This makes it very difficult to specifically reduce the levels of a particular apocarotenoid, such as b-cyclocitral, using genetic approaches.
• apocarotenoids include, but are not limited to, abscisic acid, apocarotenal, bixin, b-ionone, b-cyclocitral, crocetin, safranal, dihydro ⁇ -Ionone, dimethyl-b- cyclocitral, dihydroactinidiolide (DHAD), ethyl ester of beta-apo-8’-carotenic acid, a-ionone, pseudoionone, peridinin, apo-l0-carotenal (ApolO), apo-l2-carotenal (Apol2), apo-l4- carotenal (Apol4), apo-l4-carotenoic acid, apo-l4-carotenol, apo-l6-carotenal (Retinal), retinol, retinoic acid (RA), dehydroretinal, tretinoin, isotretinoin, alitret
• the apocarotenoid of the disclosure is selected from the group consisting of b-ionone, b-cyclocitral, safranal, dihydro ⁇ -Ionone, dimethyl ⁇ -cyclocitral, dihydroactinidiolide (DHAD), a-ionone, pseudoionone, apo-l0-carotenal, apo-l2-carotenal, apo-l4-carotenal, apo-l6-carotenal (aka Retinal) and/or any derivatives (including functional derivatives) and/or analogues and/or combinations thereof.
• DHAD dihydroactinidiolide
• a-ionone, pseudoionone apo-l0-carotenal, apo-l2-carotenal, apo-l4-carotenal, apo-l6-carotenal (aka Retinal) and/or any derivatives (including functional derivatives) and/or analogues and/or combinations thereof.
• the apocarotenoid of the disclosure is selected from the group consisting of b-ionone, b-cyclocitral, safranal, dihydro ⁇ -Ionone, dimethyl ⁇ -cyclocitral, dihydroactinidiolide (DHAD), a-ionone, pseudoionone, apo-l0-carotenal, apo-l2-carotenal, apo-l4-carotenal, apo-l6-carotenal (Retinal), and combinations thereof.
• DHAD dihydroactinidiolide
• a-ionone, pseudoionone apo-l0-carotenal, apo-l2-carotenal, apo-l4-carotenal, apo-l6-carotenal (Retinal), and combinations thereof.
• the apocarotenoid is an apocarotenal.
• the apocarotenoid is a hydrophobic carotenal (e.g., ApolO, Apol2, Apol4, and Retinal) or a hydrophilic carotenal (e.g., Beta-ionone and Beta-cyclocitral).
• the apocarotenoid of the disclosure is selected from the group consisting apo-lO-carotenal, apo-l2-carotenal, apo-l4-carotenal, apo-l6-carotenal (aka Retinal), or a combination thereof.
• the apocarotenoid of the disclosure is apo-l4-carotenal or retinal. Apo-l4-carotenal and retinal are particularly useful in methods to improve growth of the plant. In other embodiments, the apocarotenoid of the disclosure is apo-l4-carotenal. In other embodiments, the apocarotenoid of the disclosure is retinal.
• a functional derivative of the present disclosure can be a compound that is derived from an apocarotenoid and has a similar activity to the apocarentoid.
• Examples of functional derivatives of an apocaretnoid include, but are not limited to, oxidized (retinoic acid) and reduced (retinol) forms of retinal and apo-l4-carotenal.
• the apocarotenoid of the disclosure is present in the composition in a concentration of about 0.01 mM to about 100 mM.
• the apocarotenoid of the disclosure is present in the composition in a concentration of about 0.01 pM to about 50 mM, or about 0.01 pM to about 10 mM, or about 0.01 pM to about 5 mM, or about 0.01 pM to about 2 mM, or about 0.01 pM to about 1 mM, or about 0.01 mM to about 500 mM, or about 0.1 mM to about 100 mM, or about 0.1 mM to about 50 mM, or about 0.1 mM to about 50 mM, or about 0.1 mM to about 10 mM, or about 0.1 mM to about 5 mM, or about 0.1 mM to about 2 mM, or about 0.1 mM to about 1 mM, or about
• the apocarotenoid of the disclosure is present in the composition in a concentration of about 0.01 mM to about 1 mM, or about 0.01 mM to about 500 mM, or about O.l mM to about 100 mM, or about 0.05 mM ⁇ o 0.15 mM, or about 50 mM ⁇ o about 150 mM, or about 5 mM to about 15 mM. In certain embodiments, such concentrations are useful for improving plant growth. In certain embodiments, the concentration of about 0.05 mM to 0.15 mM is useful in improving the growth of Arabidopsis. In certain
• the concentration of about 0.01 mM to 100 mM is useful in improving the growth of maize, cucumber, sunflower, rice, and carrot roots.
• the apocarotenoid of the disclosure is present in the composition in a concentration of more than about 1 mM, or more than about 10 mM, or more than about 20 mM, or about 1 mM to about 10 mM, or about 1 mM to about 20 mM, or about 1 mM to about 50 mM, or about 5 mM to about 10 mM, or about 5 mM to about 15 mM, or about 5 mM to about 20 mM, or about 10 mM to about 100 mM, or about 10 mM to about 20 mM.
• concentrations are useful for reducing plant growth.
• compositions of the disclosure can further comprise one or more substances formulated for at least one agricultural application.
• the compositions of the disclosure further comprise one or more of agriculturally acceptable carrier, excipient, and/or diluent.
• agriculturally acceptable carrier e.g., calcium, nitrogen, potassium, phosphorous
• micronutrients e.g., metal ions
• insecticides fungicides, nematocides, bactericides, acaricides, herbicides, plant nutrients, rooting stimulants, chemosterilants, semiochemicals, repellents, attractants, pheromones, feeding stimulants, microbial inocula or entomopathogenic bacteria, viruses, fungi, and other signal compounds including apocarotenoids, flavonoids, jasmonates and strigolactones (Akiyama et al., Nature , 2005, 435:824-827; Harrison, Ann.
• fertilizers e.g., calcium, nitrogen, potassium, phosphorous
• Agricultural applications are understood to include, but not be limited to, yield improvement, pest control, disease control, weed control, and resistance to abiotic environmental stress.
• compositions described herein can be produced by known processes, for example, as a formulation of one or more of the compounds described herein.
• the compounds may be optionally mixed with further active ingredients, additives and/or customary formulation auxiliaries, which are then applied in a customary manner, such as, diluted with water, or as what are called tank-mixes by co- dilution of the separately formulated or partially separately formulated component(s) with water.
• the compositions are formed in a dry mixture that is added to the soil in, on and/or around the plant. Such formulations may dissolve when exposed to water and thereby be taken up by the leaves and/or roots of the plant.
• the application at different times split application
• the separately formulated or partially separately formulated one or more compounds e.g., the application of an apocartoid followed by an additional component, e.g., herbicide, fungicides, etc.
• additional component e.g., herbicide, fungicides, etc.
• the growth regulators described herein have been found to be able to modify root architecture when applied exogenously to a plant, such as Arabidopsis thaliana. Some such modifications relate to the lateral roots of a plant, which constitute the majority of a mature plant’s root system and are vital for plant anchorage, absorption of water and nutrients, and survival in the presence of diverse environmental stresses such as drought.
• one aspect of the disclosure provides methods for regulating growth in a plant, the method comprising exogenously contacting a composition comprising an effective amount of one or more apocarotenoids to the plant, a plant part, or a plant seed.
• the plants may be regulated to improve growth of the plant.
• the growth may be improved by modifying the root architecture of the plant.
• the growth also may be improved by altering (e.g., improving) lateral root formation or lateral root branching in the plant.
• the lateral roots of a plant which constitute the majority of a mature plant’s root system, are vital for plant anchorage, absorption of water and nutrients, and survival in the presence of diverse environmental stresses such as drought.
• the growth of the plant is improved by at least about 10% compared to the plant not contacted with the composition of the disclosure.
• the growth of the plant is improved by at least about 12%, or by at least about 15%, by at least about 17%, or by at least about 20%, or by at least about 25%, or by at least about 30%, or by at least about 40%, or by at least about 50%, or by at least about 75%, or even by at least 100% or more as compared to the plant not contacted with the composition.
• the plants may be regulated to reduce growth of the plant.
• This method may be particularly useful in controlling unwanted plants, such as weeds, that grow among plant crops (i.e., act as herbicides).
• the growth may be reduced by modifying the root architecture of the plant.
• the growth of the plant is reduced by at least about 20% compared to the plant not contacted with the composition of the disclosure.
• the growth of the plant is reduced by at least about 25%, or by at least about 30%, or by at least about 40%, or by at least about 50%, or by at least about 75%, or even by at least 100% or more as compared to the plant not contacted with the composition.
• One aspect of the disclosure provides methods for improving drought tolerance in a plant, the method comprising exogenously contacting a composition comprising an effective amount of one or more apocarotenoids to the plant, a plant part, or a plant seed.
• the improvement of drought tolerance is essential for stable and adequate crop production in drought-prone areas.
• Recent studies have determined that alteration of root system architecture affects (e.g., improves) drought tolerance (Uga et al., Nature Genetics , 2013,
• composition comprising an effective amount of one or more apocarotenoids, such as retinal, improves plant’s root systems by, for example, making them deeper and more highly branched, which enhances drought tolerance.
• One aspect of the disclosure provides methods for fertilizing plant soil, the method comprising providing a composition comprising an effective amount of one or more apocarotenoids to the soil.
• the composition is applied to the plant, a plant part, a plant seed, and/or an area in a low concentration (e.g., in the nM to mM concentration ranges).
• high doses of apocarotenoids can act as herbicides.
• yet another aspect of the present disclosure provides a method for controlling a harmful or unwanted plant in a crop comprising, consisting of, or consisting essentially of applying a herbicidally active amount of a composition as described herein to the harmful plant, a part of the harmful plant, a seed of the harmful plant, and/or an area in which the harmful plant grows (e.g., a cultivation area for a crop).
• a harmful or unwanted plant include, but are not limited to, a weed, an invasive species of plant (e.g., English Ivy, Kudzu), or a poisonous plant (e.g., poison oak, poison ivy).
• the composition disclosed herein is contacted with the plant. Any part of the plant can be contacted with the compositions of the disclosure, including tubers, roots, stems, leaves, flowers, and fruits.
• the composition can be applied directly to seeds or plants, or can be placed in soil in the vicinity of a seed or plant prior to or at the time of planting.
• the composition is sprayed on seeds, tubers, or foliage. Seedlings, as well as more mature plants, can be treated. Flowers and fruits can also be treated by spraying. Roots of transplants can be sprayed or dipped in the composition prior to planting.
• the composition of the disclosure may applied, for example, by contacting the unwanted plants (for example harmful plants such as mono- or dicotyledonous weeds or unwanted crop plants), the seed (for example grains, seeds or vegetative propagation organs such as tubers or budded parts of shoots), or the area on which the plants grow (for example the area under cultivation).
• the composition can be applied to the plant, a plant part, a plant seed, and/or an area in a high concentration such that the growth of harmful or unwanted plants are controlled, reduced, prevented, or eliminated.
• composition of the disclosure, and any additional compounds can be deployed together (for example as a ready-made formulation or by the tank-mix method) or successively in any sequence, for example by application by irrigating, spraying, watering and sprinkling, or by granule scattering, or by soil injection.
• the amounts of the composition (and any additional compounds) which are optimal in each case depend on the nature of the composition used and any additional compounds used and on the nature and development stage of the plant stock to be treated, and can be determined in each individual case by simple, routine preliminary tests.
• compositions of the disclosure can be used for pretreatment of the seed of the crop plant (for example for dressing of the seed), or introduced into the seed furrows prior to sowing, or employed alone or together with an additional compound(s) prior to or after emergence of the plants.
• Pre-emergence treatment includes both the treatment of the area under cultivation (including any water present in the area under cultivation, for example in the case of applications to rice) prior to sowing and the treatment of the area under cultivation in which seeds have been sown but which is not yet covered by growing plants.
• composition of the disclosure can be applied directly to the plant.
• compositions of the disclosure and any additional compounds are applied directly to the roots of the plant.
• the compositions of the disclosure and any additional compounds are applied directly to soil around the plant (for example, by soil injection).
• the compositions of the disclosure and any additional compounds are dissolved in water and applied directly to the plant and/or soil of the plant.
• contacting the composition to the plant is accomplished, for example, by irrigation, watering, sprinkling, spraying, or broadcasting.
• the formulations in commercial form are, if appropriate, diluted in a customary manner, for example in the case of wettable powders, emulsifiable concentrates, dispersions and water-dispersible granules with water.
• Dust-type formulations, granules for soil application or granules for broadcasting, and sprayable solutions are not normally diluted further with other inert substances prior to application.
• the composition of the disclosure can be applied to monocot or dicot plants, and to legumes and non-legumes. In one embodiment, the composition is applied to field-grown plants. In another embodiment, the composition is applied to greenhouse-grown plants. In one embodiment, for example, the composition can be applied to tomatoes, rice, corn, cotton, canola, wheat, barley, sugar beet, turf grass, soybeans, peas, chickpeas, dry beans, peanuts, clover, or alfalfa.
• Yet another aspect of the present disclosure provides a method for identifying a plant protein associated with a root development phenotype, the method comprising identifying a plant protein that has greater than about 40% to about 90% homology to a vertebrate retinoid binding protein, and determining whether one or more mutations in a gene encoding the plant protein results in a root development phenotype as compared to a wild type plant.
• the plant protein has about 40%, 41%, 42%, 43%, 44% 45%, 46%, 47%, 48%, 49% 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% homology or greater to the vertebrate retinoid binding protein. In certain embodiments, the plant protein has greater than about 70%, 75%, 80%, 85%, or 90% homology to the vertebrate retinoid binding protein.
• the term“significant homology” as used herein can be an Expect (E) value of less than about 5. Protein sequences having significant homology can range from about 40% to about 90% or more homology. Homology between two proteins can be determined using methods known in the art (e.g., Basic Local Alignment Search Tool (BLAST)).
• BLAST Basic Local Alignment Search Tool
• root development phenotype refers to the observable properties of root growth of the plant.
• a root development phenotype can be associated with one genotype or multiple genotypes in the plant.
• a genotype associated with a root development phenotype can include mutations in putative plant RBP-encoding genes.
• a root development phenotype can also be the result of increased or decreased expression of a protein in part of a plant (e.g., the meristem).
• a mutant genotype can be identified as associated with a root development phenotype by comparing the root growth of a plant containing the mutant genotype to the root growth a plant not containing the genotype (e.g., a wild-type plant). In some
• the mutant genotype is a non-naturally occurring mutant genotype.
• a root development phenotype can also be associated with environmental factors.
• a root development phenotype can exhibit reduced or enhanced root development.
• Examples of a root development phenotype include, but are not limited to, reduced lateral root branching, reduced lateral root formation, reduced cell elongation, short root meristems, increased lateral root branching, increased lateral root formation, increased cell elongation, and long root meristems.
• the plant protein associated with a root development phenotype can have one or more mutations (e.g., 1, 2, 3, 4, 5 or more).
• Arabidopsis thaliana All seeds were in the Columbia-0 background. Seeds were sterilized with chlorine gas, incubated in 4 °C water for 2 days, and plated on 1% (wt/vol) Murashige and Skoog (1% MS) media with 1% agar. They were exposed to 100-130 pmol/(m 2 s 1 ) illumination and grown vertically under long-day conditions at 22 °C. Plants were analyzed at 7 days after stratification (das), unless noted otherwise.
• Dl5-treated media was made using D15 (/V-(4-fluorobenzyl)-methoxy cinnamic hydroxamic acid) synthesized by LeadGen Labs, LLC solubilized in DMSO. 100 mM D15 stock solution was diluted directly into 1% MS media for final concentrations ranging from 1 mM to 100 mM.
• Example 1 Role of the carotenoid pathway in regulating lateral root development
• apocarotenoid synthesis in Arabidopsis leads to defects in de novo root organogenesis, called lateral root organogenesis. Although there are several well- characterized apocarotenoid regulators of certain developmental processes in plant roots, none of these molecules could rescue post-embryonic root organogenesis upon inhibition of carotenoid metabolism. This suggests that a previously uncharacterized apocarotenoid is essential for post-embryonic root organogenesis in Arabidopsis. Because retinoids are crucial regulators of development in vertebrates and are also known to bind to photosensitive proteins across different domains of life, it was hypothesized that retinoids could function in root development.
• FIG. 1A One advantage of studying developmental biology using the root as a model system is that spatial regions of the root have distinct developmental features (FIG. 1A).
• the meristem which is comprised of actively dividing pluripotent cells, is located at the tip of the root. As meristematic cells are displaced from the root tip, they lose their mitotic activity and begin lengthening in a region called the elongation zone. Once cells reach their full length, they acquire their final mature features in the differentiation zone. This generates a developmental gradient from pluripotency to differentiation that can be imaged along the length of the root.
• RBP retinal binding protein
• Example 2 An RBP reporter reveals dynamic retinoid binding activity in roots
• FIG. IB retinal binding proteins
• merocyanine aldehyde fluoresces in a dynamic manner in distinct developmental regions in the root (FIG. 1C).
• Merocyanine aldehyde has fairly constant fluorescence intensity in the root meristem and has intense temporal pulses of fluorescence in the elongation and differentiation zones. These spatial and temporal fluorescence patterns suggest that proteins in the root have the ability to bind to retinal endogenously.
• retinal has been identified as a chemical component of several plant species (Fleshman, et al., J Agric Food Chem , 2011, 59(9):4448-54; Giuffrida, et al., Food Chem , 2017, 231 :316-323), it has not been found in Arabidopsis.
• an extraction, chemical derivatization, and HPLC-MS analysis protocol was developed to detect hydrophobic apocarotenoids in plant tissue. Retinal was identified, as well as three other related apocarotenoids in Arabidopsis tissue (FIG. 2A).
• D15 an inhibitor of carotenoid cleavage dioxygenase activity previously shown to prevent proper root growth and lateral root organogenesis.
• D15 treated plants levels of retinal as well as the related apocarotenoids were reduced (FIG. 2B).
• D15 did not affect levels of more hydrophilic apocarotenoids such as beta-cyclocitral and beta-ionone.
• the first known stage of lateral root organogenesis is the oscillation of expression of certain genes, which is called the lateral root clock.
• D15 has been shown to reduce the amplitude of oscillations in a reporter for the lateral root clock (pDR5:LUC).
• Lateral roots constitute the majority of a mature plant’s root system and are vital for plant anchorage, absorption of water and nutrients, and survival in the presence of diverse environmental stresses such as drought tolerance.
• D15 works by preventing carotenoid cleavage and blocking the formation of apocarotenoids. Inhibition of apocarotenoid formation leads to decreased lateral root initiation.
• the initiation of lateral roots can be observed using the pDR5:LUC marker line, which oscillates in the region of lateral root initiation. The oscillation maxima determines the new site for a lateral root to initiate. In Dl5-treated plants, the amplitude of oscillations is significantly decreased (FIG. 3).
• Apo-lO-carotenal, apo-l2-carotenal, apo-l4-carotenal, and apo-l6-carotenal (retinal) all show the ability to act as plant growth enhancers, and these apocarotenoids can present new ways to stimulate crop growth and protect crops from environmental stress.
• Example 5 Plant RBP homologues are important for proper lateral root organogenesis
• Table 1 Putative RBP homologues in plants.
• RBP homologues 11 have been identified as being enriched in the root meristem in previously published tissue-specific RNA-seq experiments (Song, L.; Yamada, M. et al. Developmental Cell, 2016 and Brady, S. M.; Orlando, D. A. et al. Science 2007) or in microscopy experiments using transgenic reporters (Tejos et al., J Cell Sci , 2018, 131(2)).
• Single mutants for seven of these proteins have been tested for defects in lateral root branching, lateral root organogenesis, and meristem size.
• RBPH mutants were characterized for defects in lateral root development.
• CRINKLY-related 3 (CCR3), a protein that has been previously implicated as a suppressor in lateral root development (De Smet et al., Science , 2008, 322(590l):594-7), was found to have 80% homology to RA binding proteins in vertebrates. These measurements confirm that mutations in CCR3 result in increased lateral root formation. Although a root development phenotype was not observed in the single PATELLIN3 mutant, quadruple mutants of PATELLIN proteins have been shown to have abnormally short root meristems (Tejos et al., ./ Cell Sci , 2018, 131(2)). Overall, these results indicate that six of the seven RBPH protein families examined have significant defects in Arabidopsis root development. This further suggests that endogenous RA binds to proteins in Arabidopsis and that this interaction may be important for root development.
• Example 6 RBP activity can be observed in crop species
• Retinal may be a precursor to an active retinoid-related compound
• retinoids play an important role in plant development.
• retinoic acid is the predominant developmentally active retinoid compound, although RA and retinol also have physiological functions.
• mammalian RBPs have been shown to bind to retinal-related compounds, including Apol4, although there is no known function of Apol4 in mammals (Eroglu et ah, JBC, 2012, 287(19): 15886-95). Which apocarotenoid compounds are the primary active signaling molecules in plant root development remains unknown.
• the fluorescence intensity profile of merocyanine aldehyde indicates that proteins in plants can bind retinal, since oxidized and reduced merocyanine aldehyde (which respectively report retinoic acid and retinol binding) have different fluorescence excitation and emission maxima. However, either of these molecules may still be active in plant roots. In addition, the other hydrophobic retinal-related apocarotenoids, such as Apol4, could be the predominant active compound in plants.
• Apol4 hydrophobic retinal-related apocarotenoids
• One feature of the retinal-related compounds tested for their ability to affect root development is that they can all be metabolized to retinal. This suggests that retinal is the most probable precursor to the active metabolite, or the active metabolite itself.
• apocarotenoid compounds were identified. Partially specific inhibition of retinal and related compounds by D15 results in defects in root growth and lateral root organogenesis. Addition of retinal or Apol4 fully rescued these defects. These results indicate that retinal or Apol4 is either the precursor or active signaling molecule essential for root growth and branching.
• the presence of plant proteins with homology to vertebrate RBPs further suggests that retinoids are capable of interacting with plant proteins.
• characterization of these plant RBP homologues revealed that retinoids and their binding partners are essential regulators of root development. The ability of these apocarotenoids to alter root development through exogenous application supports their potential use to enhance root branching and growth in crop plants

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